Super-Pure RNA Extraction Kit (TRIzol)

1. Компоненты набора реагентов

2. Хранилище

Этот набор реагентов следует хранить при комнатной температуре. (15-25℃) in a dry environment and is stable for 12 месяцы. DNase I contains a preservative, возможность транспортировки при комнатной температуре, но для длительного хранения, it should be kept at -20℃.

3. Инструкции по использованию набора реагентов

3.1 This kit is intended for molecular biology research purposes and should not be used for disease diagnosis or treatment.

3.2 Some components in the kit contain irritants; it is advisable to take necessary precautions (such as wearing protective clothing and goggles).

3.3 The use of this kit requires additional equipment such as a high-speed centrifuge, водяная баня (металлическая ванна), вихревой смеситель, безводный этанол, liquid nitrogen, chloroform, стерильная деионизированная вода, and EP tubes.

4. Знакомство с набором реагентов

This RNA purification kit utilizes the traditional TRIzol combined with column membrane method for the rapid purification of plant, животное, салфетка, клетка, microbial RNA, and fungal hyphae RNA. It is suitable for most species. This RNA purification kit can be applied to plant tissues exceeding 100 мг. The RNA extracted with this kit has extremely low DNA content. If sensitivity to DNA in experiments is a concern, it is recommended to use DNase I digestion on the column.

The RNA Fast Purification Kit can extract total RNA from samples (including nuclear RNA and cytoplasmic RNA) в пределах 1 час. The extracted RNA can be directly used for RT-ПЦР, Нозерн-блоттинг, и другие приложения.

5. Экспериментальные принципы и процедуры

6. Процесс экстракции

Precautions before starting the experiment:

А. Перед использованием, add the specified amount of absolute ethanol to СтиратьБуфер 1 according to the label on the reagent bottle, and check the box on the label to indicate that absolute ethanol has been added.

Б. Элюирующий буфер представляет собой 0.1х ТЕ решение containing minimal EDTA. If EDTA affects subsequent experiments, it is recommended to replace the Elution Buffer with sterile deionized water.

1. Образец обработки:

А. Салфетка: If fresh materials cannot be used immediately, place them in liquid nitrogen and store them at -80°C. Dried materials can be stored at room temperature. Grind 30~80 mg of tissue in liquid nitrogen and add 1 ml Trizol buffer for homogenization.

Б. Monolayer Cultured Cells: Aspirate the culture medium and add 1 ml Trizol buffer for homogenization.

С. Cell Suspension: Centrifuge to collect cells and add 1 ml Trizol buffer for homogenization.

2. After adding Trizol buffer к образцу, mix thoroughly by pipetting, allow complete sample lysis, and incubate at room temperature for 5 минуты.

3. Add chloroform in a ratio of 200 μl per 1 ml of Trizol buffer, vigorously shake for 30 секунды, and incubate at room temperature for 2 минуты.

4. Centrifuge the lysate at 4°C, 12,000 об/мин для 10 минуты. RNA will be present in the upper aqueous phase.

5. Carefully transfer the supernatant to a new centrifuge tube, avoiding the collection of the middle and bottom layers. (Примечание: Approximately 400 μl of liquid can be transferred, which may be less for some species.)

6. Add an equal volume of pre-chilled absolute ethanol, mix quickly. (Например, добавлять 400 μl of lysate, добавлять 400 μl of absolute ethanol. If the lysate volume is less than 400 мкл, reduce the amount of absolute ethanol proportionally. A slight precipitate may form after adding ethanol, but it does not affect subsequent experiments.)

7. Transfer the obtained liquid to an RNA purification column (approximately 650-700 μl per time), centrifuge at more than 8,000 об/мин для 1 минута, discard the collected waste, and reinsert the collection tube into the purification column for the next step.

8. Повторить шаг 7, adding the remaining liquid to the RNA purification column, centrifuge at more than 8,000 об/мин для 1 минута, discard the waste and the collection tube.

9. Place the RNA purification column in a new collection tube, добавлять 300 μl of Inhibitor Removal Buffer, centrifuge at more than 8,000 об/мин для 1 минута, выбросить отходы, and reinsert the RNA purification column into the tube for the next step.

10. Добавлять 80 μl of DNase Iworking solution to the RNA purification column, incubate at 20°C to 30°C for 15 минуты. (Prepare DNase I working solution: 62 μl RNase-Free Water, 8 μl 10× Reaction Buffer, и 10 μl DNase I, make up to 80 μl DNase I working solution.)

11. Добавлять 300 μl of Inhibitor Removal Bufferto the RNA purification column, centrifuge at more than 8,000 об/мин для 1 минута, выбросить отходы, and reinsert the RNA purification column into the tube for the next step.

12. Добавлять 700 мкл промывочного буфера 1to the RNA purification column, центрифуга в 14,000 об/мин (20,000×г) для 2 минуты, extend the centrifugation time if needed for a drier membrane. (Примечание: Confirm the addition of ethanol to Wash Buffer 1; ethanol presence significantly affects subsequent experiments. Ensure the membrane is dry after centrifugation before elution. Discard the waste and the collection tube. After using Wash Buffer 1, the membrane on the RNA purification column should only have a slight color. Carefully remove the RNA purification column after centrifugation, ensuring it does not touch the collection tube to avoid ethanol contamination.)

13. Place the RNA purification column in a new centrifuge tube, drip 100 мкл элюирующего буферана мембрану, incubate at room temperature for 5 минуты (15°C to 25°C), and centrifuge at more than 8,000 об/мин для 1 минута. (Примечание: Eluting RNA with 50 μl of Elution Buffer can increase RNA concentration but decrease total RNA yield.)

14. Repeat the previous step. (Примечание: A new centrifuge tube can be used to collect the RNA eluted the second time or continue using the original collection tube to collect RNA.)